AAC chap 3 principles of electrophoresis new

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Chap 3 Electrophoresis Methods
Biochemistry and Molecular Biology
10.1 general principles
10.2 Support media
10.3 Electrophoresis of protein
10.4 Electrophoresis of nucleic acids
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Principle of Electrophoresis (電泳)
v = m ´ E
v = migration velocity of charged species (cm/s)
m = electrophoretic mobility (cm
2
/V´s)
charge, size, shape of molecule,
viscosity, pore size, buffer pH and
ionic strength, temperature of medium
E = electric field strength (Volt/cm)
-
Migration of charged particles in an electric field
1. Free solution
2. Stable media
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Arne Tiselius
(Sweden, 1902-1971)
The Nobel Prize in Chemistry 1948
This type of cell is essentially a bent glass tube with
electrolyte reservoirs containing the cathode and
anode, and a buffer containing the macromolecules
that need electrophoresed.
He tested horse serum in the apparatus and found 4

distinct bands consisting of albumin and 3 globulin
components, which he named “α,” “β,” and “γ.”
"for his research on electrophoresis and adsorption
analysis, especially for his discoveries concerning the
complex nature of the serum proteins"
Father of Electrophoresis
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Gel Electrophoresis (電泳)
most common method of separation in biological lab.
材料: Gel made of agarose
or polyacrylamide
對象：Protein = 30-50 kD
DNA = >2000 kD
+
–
原理: Separation is based upon the mobility
of charged macromolecules under the
influence of an electric field.
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Where are the charges from? - Proteins
Charged side chain
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Where are the charges from? —Protein
胺基酸帶有可解離的胺基(-NH
3
+
)和羧基(-COO-)，，是典型的兩性
電解質
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Where are the charges from? DNA

DNA : Backbone
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＋
－
Capillary wall
Applied electric field, E=applied voltage /length of support medium
Electroosmotic Flow (EOF)
Salts, buffer and ion
Cathode
Anode
Acidic silanol groups impart negative charge on wall
Electrolyte cations are attracted to the capillary walls, forming
electrical double layers. When a voltage is applied, the net movement
of the electrolyte solution towards the cathode is known as
electrophoresis.
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Cathode
Anode
EOF
−
+
Electroosmosis: bulk liquid migrates relative to the stationary
solid phase because of the imposed electric field
Electroosmotic Flow (EOF): occurs due to charge on capillary
walls above pH 3 under electric field
SiOH → SiO
−
+ H pKa ≈ 6
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v[M
-
] < v[M], < v[M
+
]
CathodeAnode

EOF
V
electroosmotic
v=v
electroosmotic (EOF)
+ v
electrophoretic
V
electrophoretic + +
+
−
−
−
+ +
+
−
−
−
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Factors Affecting Electrophoresis
Electrophoretic velocity depends on:
Inherent Factors External Environment
 Magnitude of its charge
 Charge density
 Molecular weight
 Tertiary or quaternary
structure (i.e., its shape).
 Solution pH
 Electric field
 Solution viscosity

 Temperature
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毛細管區帶電泳
線上偵測方式：
（1）螢光
（2）紫外-可見光
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–
=
–
+
=
++
+
++
NegativeNeutral Positive
–
=
–
+ =++ +
++
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Electrophoretic Methods
Zone Electrophoresis Isotachophoresis Isoelectric Focusing
homogeneous buffer
discontinuous
buffer system
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Polyacrylamide Gel
BMB10.2.1

Acrylamide
(major, polymerization)
2. Head to tail polymerization
1. Free radical catalysis
S
2
O
8
2-
+ e
-
SO
4
-
+SO
4
-
x
TEMED
TEMED
N, N, N’,N’-
tetramethylenediamine
SO4-x
SO4-x SO4-x SO4-x
etc.
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Polyacrylamide Gel Electrophoresis (PAGE)
Acrylamide
(major, polymerization)
Methylene bis-Acrylamide

(minor, for cross-linking)
Methylene
Head to tail polymerization
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Formation of Polyacrylamide Gel
S
O
4
-
x
S
O
4
-
x
SO4-x
SO4-x
SO4-x
The pore size of the gel varied by changing the
concentration of both acrylamide and bis-acrylamide
Polymerization
Cross-linking
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Sodium Dodecyl Sulphate
 Solubilize protein
 Add negative charge to protein
S
S

- - - - -

- - - -
SDS binds to protein homogeneously and
stoichiometrically
1 SDS – 2 amino acids residues
S
S
-
-
-
-
-
S
S
SS
SS





-
Add SDS
1. Disulfide bond breakage
(by mercaptoethano)
2. Denature protein structure
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Polyacrylamide Gel Electrophoresis (PAGE)
Buffer
Solution (−)

Buffer Solution (+)
Stacking Gel
 Large pore size
 Concentrate protein
4% acrylamide. pH 6.8
(Tris-buffer, glycine-slow, tailing ion)
Separation Gel
5-20 % acrylamide. pH 8.8
(Tris, HCl-Cl
-
, fast ion)
S
S
-
-
-
-
-
S
S
SS
SS





1. Proteins (negatively charged due to SDS) move to positive
electrode
2. Proteins separate by size

3. Smaller proteins move faster
Glycine, pK1:2.34, pK2, 9.4)
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%T : Solids content
%C : Ratio of cross-linker to acrylamide monomer
The pore size may be controlled by varying total concentrations
of monomer and cross-linker, and by varying their ratio.
5 %T
10 %T 15 %T 20 %T
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Polyacrylamide Gel Electrophoresis (PAGE)
Molecular sieving properties of the gel
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Polyacrylamide Gel Electrophoresis (PAGE)
 Separation of protein by Size
 Purification of Proteins
 Estimation of protein molecular weight
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Visualizing of Separated Protein Band
Protein Detection Methods
 Coomassie Blue 0.1 mg/band - 1 mg/band
 Colloidal Coomassie 10 - 100 ng/band
 Reverse Staining with
 Metal Ion 10 - 100 ng/band
 Fluorescent Stain 1 - 10 ng/band
 Silver Stain 1 - 10 ng/band
 1 ng of a 10kDa 100 femtomoles
 1 ng of a 100kDa 10 femtomoles
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Protein

Acidic Neutral
Basic & Aromatic
Side chains
Coomassie Blue
Coomassie Brilliant Blue
R-250
e.g. Arginine
Two prinicples
1. -SO3
2–
react with
positive residues
2. Benzene react with
hydrophobic resides
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Molecular Weight of Standards
Size of proteins in Kaleidoscope
standard is known
Plot Distance Migrated (mm) vs.
Size (kDa) on semilog graph paper